Abstract: Cryptococcus neoformans is a pathogenic fungus that is found world-wide and causes meningioencephalitis, particularly in immunocompromised individuals. It is invariably fatal unless treated, and the current antifungals are inadequate to effectively cure this disease, due to inherent toxicities, the inability to kill the fungus and prevent relapse, or innate resistance to the class of antifungals. Recent studies have indicated that there are over 1,000,000 new cases of cryptococcosis in the world each year, which results in over 600,000 deaths. New agents to treat Cryptococcus are needed, and the fungal cell wall is an attractive target, since it is unique to fungi and absent in humans. Echinocandins are a class of antifungals that target glucan synthesis in the cell wall. Cryptococcus is naturally resistant to the echinocandins. We have shown that disruption of the cell wall integrity (CWI) signaling pathway causes Cryptococcus to become highly sensitive to echinocandins, and identified candidate transcription factors that may play a role in echinocandin resistance. This pathway is the major pathway that impacts cell wall and is dependent on protein kinase C (Pkc1). The CWI/PKC1 pathway plays a key role in response to heat shock, oxidative and nitrosative stress, and cell wall inhibitors. Our preliminary data suggests that this pathway is part of a highly connected network that controls cell wall biosynthesis, repair and remodeling. The major goal of this project is to define the mechanism(s) by which cell wall integrity is maintained in response to specific cell wall inhibitors, including echinocandins and to identify potential gene targets for antifungals with synergy to echinocandins. There are three specific aims: In the first aim, we will define the role of the cell wall integrity pathway in echinocandin resistance using genomic and network analysis approaches. In the second aim, we will identify the genes essential to cell wall integrity and that contribute to echinocandin resistance by screening large deletion sets for specific phenotypes. In the third aim, we will assess the functional interactions of the cell wall integrity components. In this highly collaborative project, we propose to use our complementary expertise to integrate genetic, phenotypic and biochemical data to identify and characterize the network of interactions governing cell wall homeostasis in C. neoformans. This will significantly advance our understanding of cell wall maintenance and remodeling in a human pathogen and will provide multiple targets for designing specific antifungal drugs that are less likely to be toxic in mammals.